Ribaxamase, an Orally Administered ß-Lactamase, Diminishes Changes to Acquired Antimicrobial Resistance of the Gut Resistome in Patients Treated with Ceftriaxone.

INTRODUCTION: Intravenous (IV) ß-lactam antibiotics, excreted through bile into the gastrointestinal (GI) tract, may disrupt the gut microbiome by eliminating the colonization resistance from beneficial bacteria. This increases the risk for Clostridium difficile infection (CDI) and can promote antimicrobial resistance by selecting resistant organisms and eliminating competition by non-resistant organisms. Ribaxamase is an orally administered ß-lactamase for use with IV ß-lactam antibiotics (penicillins and cephalosporins) and is intended to degrade excess antibiotics in the upper GI before they can disrupt the gut microbiome and alter the resistome. METHODS: Longitudinal fecal samples (349) were collected from patients who participated in a previous Phase 2b clinical study with ribaxamase for prevention of CDI. In that previous study, patients were treated with ceftriaxone for a lower respiratory tract infection and received concurrent ribaxamase or placebo. Extracted fecal DNA from the samples was subjected to whole-genome shotgun sequencing and analyzed for the presence of antimicrobial resistance (AMR) genes by alignment of sequences against the Comprehensive Antibiotic Resistance Database. A qPCR assay was also used to confirm some of the results. RESULTS: Database alignment identified ~1300 acquired AMR genes and gene variants, including those encoding ß-lactamases and vancomycin resistance which were significantly increased in placebo vs ribaxamase-treated patients following antibiotic exposure. qPCR corroborated the presence of these genes and supported both new acquisition and expansion of existing gene pools based on no detectable copy number or a low copy number in pre-antibiotic samples which increased post-antibiotics. Additional statistical analyses demonstrated significant correlations between changes in the gut resistome and clinical study parameters including study drug assignment and ß-lactamase and vancomycin resistance gene frequency. DISCUSSION: These findings demonstrated that ribaxamase reduced changes to the gut resistome subsequent to ceftriaxone administration and may help limit the emergence of AMR.

Leveraging Human Microbiome Features to Diagnose and Stratify Children with Irritable Bowel Syndrome.

Accurate diagnosis and stratification of children with irritable bowel syndrome (IBS) remain challenging. Given the central role of recurrent abdominal pain in IBS, we evaluated the relationships of pediatric IBS and abdominal pain with intestinal microbes and fecal metabolites using a comprehensive clinical characterization and multiomics strategy. Using rigorous clinical phenotyping, we identified preadolescent children (aged 7 to 12 years) with Rome III IBS (n = 23) and healthy controls (n = 22) and characterized their fecal microbial communities using whole-genome shotgun metagenomics and global unbiased fecal metabolomic profiling. Correlation-based approaches and machine learning algorithms identified associations between microbes, metabolites, and abdominal pain. IBS cases differed from controls with respect to key bacterial taxa (eg, Flavonifractor plautii and Lachnospiraceae bacterium 7_1_58FAA), metagenomic functions (eg, carbohydrate metabolism and amino acid metabolism), and higher-order metabolites (eg, secondary bile acids, sterols, and steroid-like compounds). Significant associations between abdominal pain frequency and severity and intestinal microbial features were identified. A random forest classifier built on metagenomic and metabolic markers successfully distinguished IBS cases from controls (area under the curve, 0.93). Leveraging multiple lines of evidence, intestinal microbes, genes/pathways, and metabolites were associated with IBS, and these features were capable of distinguishing children with IBS from healthy children. These multi-omics features, and their links to childhood IBS coupled with nutritional interventions, may lead to new microbiome-guided diagnostic and therapeutic strategies.

The NLRP3 inflammasome mediates DSS-induced intestinal inflammation in Nod2 knockout mice.

Crohn's disease (CD) is a chronic disorder of the gastrointestinal tract characterized by inflammation and intestinal epithelial injury. Loss of function mutations in the intracellular bacterial sensor NOD2 are major risk factors for the development of CD. In the absence of robust bacterial recognition by NOD2 an inflammatory cascade is initiated through alternative PRRs leading to CD. In the present study, MCC950, a specific small molecule inhibitor of NLR pyrin domain-containing protein 3 (NLRP3), abrogated dextran sodium sulfate (DSS)-induced intestinal inflammation in Nod2-/- mice. NLRP3 inflammasome formation was observed at a higher rate in NOD2-deficient small intestinal lamina propria cells after insult by DSS. NLRP3 complex formation led to an increase in IL-1ß secretion in both the small intestine and colon of Nod2ko mice. This increase in IL-1ß secretion in the intestine was attenuated by MCC950 leading to decreased disease severity in Nod2ko mice. Our work suggests that NLRP3 inflammasome activation may be a key driver of intestinal inflammation in the absence of functional NOD2. NLRP3 pathway inhibition can prevent intestinal inflammation in the absence of robust NOD2 signaling.

Re-purposing 16S rRNA gene sequence data from within case paired tumor biopsy and tumor-adjacent biopsy or fecal samples to identify microbial markers for colorectal cancer.

Microbes colonizing colorectal cancer (CRC) tumors have the potential to affect disease, and vice-versa. The manner in which they differ from microbes in physically adjacent tissue or stool within the case in terms of both, taxonomy and biological activity remains unclear. In this study, we systematically analyzed previously published 16S rRNA sequence data from CRC patients with matched tumor:tumor-adjacent biopsies (n = 294 pairs, n = 588 biospecimens) and matched tumor biopsy:fecal pairs (n = 42 pairs, n = 84 biospecimens). Procrustes analyses, random effects regression, random forest (RF) modeling, and inferred functional pathway analyses were conducted to assess community similarity and microbial diversity across heterogeneous patient groups and studies. Our results corroborate previously reported association of increased Fusobacterium with tumor biopsies. Parvimonas and Streptococcus abundances were also elevated while Faecalibacterium and Ruminococcaceae abundances decreased in tumors relative to tumor-adjacent biopsies and stool samples from the same case. With the exception of these limited taxa, the majority of findings from individual studies were not confirmed by other 16S rRNA gene-based datasets. RF models comparing tumor and tumor-adjacent specimens yielded an area under curve (AUC) of 64.3%, and models of tumor biopsies versus fecal specimens exhibited an AUC of 82.5%. Although some taxa were shared between fecal and tumor samples, their relative abundances varied substantially. Inferred functional analysis identified potential differences in branched amino acid and lipid metabolism. Microbial markers that reliably occur in tumor tissue can have implications for microbiome based and microbiome targeting therapeutics for CRC.

Leveraging sequence-based faecal microbial community survey data to identify a composite biomarker for colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is the second leading cause of cancer-associated mortality in the USA. The faecal microbiome may provide non-invasive biomarkers of CRC and indicate transition in the adenoma-carcinoma sequence. Re-analysing raw sequence and metadata from several studies uniformly, we sought to identify a composite and generalisable microbial marker for CRC. DESIGN: Raw 16S rRNA gene sequence data sets from nine studies were processed with two pipelines, (1) QIIME closed reference (QIIME-CR) or (2) a strain-specific method herein termed SS-UP (Strain Select, UPARSE bioinformatics pipeline). A total of 509 samples (79 colorectal adenoma, 195 CRC and 235 controls) were analysed. Differential abundance, meta-analysis random effects regression and machine learning analyses were carried out to determine the consistency and diagnostic capabilities of potential microbial biomarkers. RESULTS: Definitive taxa, including Parvimonas micra ATCC 33270, Streptococcus anginosus and yet-to-be-cultured members of Proteobacteria, were frequently and significantly increased in stools from patients with CRC compared with controls across studies and had high discriminatory capacity in diagnostic classification. Microbiome-based CRC versus control classification produced an area under receiver operator characteristic (AUROC) curve of 76.6% in QIIME-CR and 80.3% in SS-UP. Combining clinical and microbiome markers gave a diagnostic AUROC of 83.3% for QIIME-CR and 91.3% for SS-UP. CONCLUSIONS: Despite technological differences across studies and methods, key microbial markers emerged as important in classifying CRC cases and such could be used in a universal diagnostic for the disease. The choice of bioinformatics pipeline influenced accuracy of classification. Strain-resolved microbial markers might prove crucial in providing a microbial diagnostic for CRC.

Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a catastrophic disease of preterm infants, and microbial dysbiosis has been implicated in its pathogenesis. Studies evaluating the microbiome in NEC and preterm infants lack power and have reported inconsistent results. METHODS AND RESULTS: Our objectives were to perform a systematic review and meta-analyses of stool microbiome profiles in preterm infants to discern and describe microbial dysbiosis prior to the onset of NEC and to explore heterogeneity among studies. We searched MEDLINE, PubMed, CINAHL, and conference abstracts from the proceedings of Pediatric Academic Societies and reference lists of relevant identified articles in April 2016. Studies comparing the intestinal microbiome in preterm infants who developed NEC to those of controls, using culture-independent molecular techniques and reported α and ß-diversity metrics, and microbial profiles were included. In addition, 16S ribosomal ribonucleic acid (rRNA) sequence data with clinical meta-data were requested from the authors of included studies or searched in public data repositories. We reprocessed the 16S rRNA sequence data through a uniform analysis pipeline, which were then synthesized by meta-analysis. We included 14 studies in this review, and data from eight studies were available for quantitative synthesis (106 NEC cases, 278 controls, 2944 samples). The age of NEC onset was at a mean ± SD of 30.1 ± 2.4 weeks post-conception (n = 61). Fecal microbiome from preterm infants with NEC had increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes prior to NEC onset. Alpha- or beta-diversity indices in preterm infants with NEC were not consistently different from controls, but we found differences in taxonomic profiles related to antibiotic exposure, formula feeding, and mode of delivery. Exploring heterogeneity revealed differences in microbial profiles by study and the target region of the 16S rRNA gene (V1-V3 or V3-V5). CONCLUSIONS: Microbial dysbiosis preceding NEC in preterm infants is characterized by increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes. Microbiome optimization may provide a novel strategy for preventing NEC.

Composition and function of the pediatric colonic mucosal microbiome in untreated patients with ulcerative colitis.

Inflammatory bowel diseases (IBD) are chronic intestinal inflammatory disorders characterized by a complex disruption of the physiologic interaction between the host immune system and intestinal microbes precipitated by environmental factors. Numerous observations indicate the altered composition and function of the intestinal microbiome of patients with ulcerative colitis (UC), a subtype of IBD. The accuracy of these results may be limited by confounding factors, such as concurrent medication use. To address these limitations, we examined the colonic mucosal microbiome of pediatric patients with UC prior to initiating treatment. Based on bacterial 16S rRNA gene sequencing, we identified a significant decrease in the phylum Verrucomicrobia in patients with UC. At the genus level, we observed a significant decrease in the short chain fatty acid producer Roseburia. Despite these compositional changes, we did not identify inferred gene content differences between the UC and control groups. To determine if microbial taxa may be associated with clinical outcomes, we retrospectively assessed the clinical course of the UC patients. Despite similar metrics of OTU richness and diversity, multiple OTU differences were observed between patients who responded to therapy and those who did not. Our observations regarding the mucosal microbiome and the associations with differential clinical outcomes support the contributions of gut microbes to disease onset and modulation.

Role of the Gut Microbiome in Obstructive Sleep Apnea-Induced Hypertension.

Individuals suffering from obstructive sleep apnea (OSA) are at increased risk for systemic hypertension. The importance of a healthy gut microbiota, and detriment of a dysbiotic microbiota, on host physiology is becoming increasingly evident. We tested the hypothesis that gut dysbiosis contributes to hypertension observed with OSA. OSA was modeled in rats by inflating a tracheal balloon during the sleep cycle (10-s inflations, 60 per hour). On normal chow diet, OSA had no effect on blood pressure; however, in rats fed a high-fat diet, blood pressure increased 24 and 29 mm Hg after 7 and 14 days of OSA, respectively (P<0.05 each). Bacterial community characterization was performed on fecal pellets isolated before and after 14 days of OSA in chow and high-fat fed rats. High-fat diet and OSA led to significant alterations of the gut microbiota, including decreases in bacterial taxa known to produce the short chain fatty acid butyrate (P<0.05). Finally, transplant of dysbiotic cecal contents from hypertensive OSA rats on high-fat diet into OSA recipient rats on normal chow diet (shown to be normotensive) resulted in hypertension similar to that of the donor (increased 14 and 32 mm Hg after 7 and 14 days of OSA, respectively; P<0.05). These studies demonstrate a causal relationship between gut dysbiosis and hypertension, and suggest that manipulation of the microbiota may be a viable treatment for OSA-induced, and possibly other forms of, hypertension.

The Microbiome, Intestinal Function, and Arginine Metabolism of Healthy Indian Women Are Different from Those of American and Jamaican Women.

BACKGROUND: Indian women have slower arginine flux during pregnancy compared with American and Jamaican women. Arginine is a semi-essential amino acid that becomes essential during periods of rapid lean tissue deposition. It is synthesized only from citrulline, a nondietary amino acid produced mainly in the gut. The gut is therefore a key site of arginine and citrulline metabolism, and gut microbiota may affect their metabolism. OBJECTIVE: The objective of this study was to identify differences in the gut microbiota of nonpregnant American, Indian, and Jamaican women and characterize the relations between the gut microbiota, gut function, and citrulline and arginine metabolism. METHODS: Thirty healthy American, Indian, and Jamaican women (n = 10/group), aged 28.3 ± 0.8 y, were infused intravenously with [guanidino-15N2]arginine, [5,5-2H2]citrulline, and [15N2]ornithine and given oral [U-13C6]arginine in the fasting and postprandial states. Fecal bacterial communities were characterized by 16S rRNA gene sequencing. RESULTS: In the fasting state, Indian women had lower citrulline flux than did American and Jamaican women [7.0 ± 0.4 compared with 9.1 ± 0.4 and 8.9 ± 0.2 µmol ⋅ kg fat-free mass (FFM)-1 ⋅ h-1, P = 0.01] and greater enteral arginine conversion to ornithine than did American women (1.4 ± 0.11 compared with 1.0 ± 0.08 µmol ⋅ kg FFM-1 ⋅ h-1, P = 0.04). They also had lower mannitol excretion than American and Jamaican women (154 ± 37.1 compared with 372 ± 51.8 and 410 ± 39.6 mg/6 h, P < 0.01). Three dominant stool community types characterized by increased abundances of the genera Prevotella, Bacteroides, and Bacteroides with Clostridium were identified. Indian women had increased mean relative abundances of Prevotella (42%) compared to American and Jamaican women (7% and < 1%, P = 0.03) which were associated with diet, impaired intestinal absorptive capacity, and arginine flux. CONCLUSIONS: These findings suggest that dysregulated intestinal function and a unique gut microbiome may contribute to altered arginine metabolism in Indian women.

Evaluating the performance of carboxylate platform fermentations across diverse inocula originating as sediments from extreme environments.

To test the hypothesis that microbial communities from saline and thermal sediment environments are pre-adapted to exhibit superior fermentation performances, 501 saline and thermal samples were collected from a wide geographic range. Each sediment sample was screened as inoculum in a 30-day batch fermentation. Using multivariate statistics, the capacity of each community was assessed to determine its ability to degrade a cellulosic substrate and produce carboxylic acids in the context of the inoculum sediment chemistry. Conductance of soils was positively associated with production of particular acids, but negatively associated with conversion efficiency. In situ sediment temperature and conversion efficiency were consistently positively related. Because inoculum characteristics influence carboxylate platform productivity, optimization of the inoculum is an important and realistic goal.

Common mechanistic themes for the powerstroke of kinesin-14 motors.

Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.

Comparison of three screening methods to select mixed-microbial inoculum for mixed-acid fermentations.

Using a mixed culture of microorganisms, the carboxylate platform converts biomass into hydrocarbons and chemicals. To develop a method that identifies the highest performing inoculum for carboxylate fermentations, five bacterial communities were screened and ranked by three fermentation performance tests: (1) 30-day batch screen, (2) 28-day continuum particle distribution model (CPDM), and (3) 5-month continuous countercurrent fermentation trains. To screen numerous inocula sources, these tests were used sequentially in an aseptic environment. For the batch-fermentation screen, Inoculum 1 achieved the highest conversion. For the CPDM evaluation, the operating map for Inoculum 1 had the highest performance. For the continuous countercurrent fermentation, the train resulting from Inoculum 1 was among the best performers. This study suggests that the three screens are a useful and predictive method for choosing optimal inocula sources. The bacterial community with optimal performance in these three screens could be considered for use in commercial-scale fermentations.

Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.

We have used cryo-electron microscopy (cryo-EM) and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1's nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor's coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.

Correction: Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules.

[This corrects the article DOI: 10.1371/journal.pone.0053792.].

A microfluidic microbial fuel cell array that supports long-term multiplexed analyses of electricigens.

Microbial fuel cells (MFCs) are green energy technologies that exploit microbial metabolism to generate electricity. The widespread implementation of MFC technologies has been stymied by their high cost and limited power. MFC arrays in which device configurations or microbial consortia can be screened have generated significant interest because of their potential for defining aspects that will improve performance featuring high throughput characteristics. However, current miniature MFCs and MFC array systems do not support long-term studies that mimic field conditions, and hence, have limitations in fully characterizing and understanding MFC performances in varieties of conditions. Here, we describe an MFC array device that incorporates microfluidic technology to enable continuous long-term analysis of MFC performance at high throughput utilizing periodic anolyte/catholyte replenishment. The system showed 360% higher power output and 700% longer operating time when compared to MFC arrays without catholyte replenishment. We further demonstrate the utility of the system by reporting its successful use in screening microbial consortia collected from geographically diverse environments for communities that support enhanced MFC performance. Taken together, this work demonstrates that anolyte/catholyte replenishment can significantly improve the long-term performance of microfabricated MFC arrays, and support the characterization of diverse microbial consortia.

Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.

Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αß-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.

Cryo-electron tomography for structural characterization of macromolecular complexes.

Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.

GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs).

Microtubule plus-end-tracking proteins (+TIPs) localize to growing microtubule plus ends to regulate a multitude of essential microtubule functions. End-binding proteins (EBs) form the core of this network by recognizing a distinct structural feature transiently existing in an extended region at growing microtubule ends and by recruiting other +TIPs to this region. The nature of the conformational difference allowing EBs to discriminate between tubulins in this region and other potential tubulin binding sites farther away from the microtubule end is unknown. By combining in vitro reconstitution, multicolor total internal reflection fluorescence microscopy, and electron microscopy, we demonstrate here that a closed microtubule B lattice with incorporated GTPγS, a slowly hydrolyzable GTP analog, can mimic the natural EB protein binding site. Our findings indicate that the guanine nucleotide γ-phosphate binding site is crucial for determining the affinity of EBs for lattice-incorporated tubulin. This defines the molecular mechanism by which EBs recognize growing microtubule ends.

Cryo-electron tomography of microtubule-kinesin motor complexes.

Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature. The rise of cryo-electron tomography within the last few years has now opened a new avenue towards solving 3-D structures of microtubule-MAP complexes that do not form helical assemblies, most importantly for the subject here, all microtubules that exhibit a lattice seam. In addition, not all motor domains or MAPs decorate the microtubule surface regularly enough to match the underlying microtubule lattice, or they adopt conformations that deviate from helical symmetry. Here we demonstrate the power and limitation of cryo-electron tomography using two kinesin motor domains, the monomeric Eg5 motor domain, and the heterodimeric Kar3Vik1 motor. We show here that tomography does not exclude the possibility of post-tomographic averaging when identical sub-volumes can be extracted from tomograms and in both cases we were able to reconstruct 3-D maps of conformations that are not possible to obtain using helical or other averaging-based methods.